The goal of this program is to investigate the integrated action of the biosynthetic enzymes that comprise the dolichol pathway. Specifically, research will focus on the enzymes that catalyze the first seven reactions that take place on the cytosolic face of the endoplasmic reticulum (ER) membrane and lead to the biosynthesis of dolichol pyrophosphate-GIcNAc2Man5. N-linked glycosylation is an essential process in all eukaryotes, and the steps leading to the biosynthesis of nascent glycoproteins are highly conserved throughout the eukaryotic kingdom. Despite the significance of this pathway in eukaryotic cells, little is known about the structure, mechanism, and integrated function of the constituent enzymes. Studies in this program will focus on the enzymes comprising the dolichol pathway in the yeast S. cerevisiae. Progress in understanding the yeast enzymes will directly enable identification of mammalian homologs and provide insight into the dolichol pathway in humans and other mammals. The specific aims of this research program are as follows: 1.To identify, biochemically characterize, and develop homologous or heterologous expression systems for the enzymes that catalyze the sequence of seven contiguous transformations in the dolichol pathway. A biochemical approach will be adopted for the identification of the "missing" enzymes in the sequence. 2.To develop experimental approaches to investigate how substrates are transferred along the "assembly line" of enzymes in the dolichol pathway. Both in vivo and in vitro methods for evaluating the pathway and the role of the dolichol-bound substrates will be presented. 3.To investigate the substrate specificity of the enzymes catalyzing the early steps in the dolichol pathway. Specificity for both glycosyl donor and acceptor substrates will be evaluated. Specifically we are interested in evaluating whether the dolichol pathway enzymes can transfer saccharide directly to a glycopeptide acceptor. Such a finding would be of utility for the preparation of tailored glycoprotein products. 4.To carry out structural analysis of targets in the pathway. This specific aim will be carried out in collaboration with Prof. Prestegard at the University of Georgia for NMR analysis and Prof. Karen Allen in the at Boston University Medical School for X-ray analysis.